1. Field of the Invention
The present invention relates to methods and apparatus for high content screening (HCS) and high throughput screening (HTS). In particular, the present invention relates to cell activity assays (CAA) involving chemotaxis, migration, angiogenesis, growth, proliferation, and other cell activity based on, for example, morphology, shape, and movement of cells. The present invention also relates to cell activity assays involving changes internal to cells such as differentiation, alteration of metabolic rate, and movement of molecules within a cell initiated by activation of receptors in the cell membrane. The present invention also relates to cell activity assays involving the interaction of cells in response to various chemical environments, and the interaction of different cell types with one another. The present invention also relates to assays involving the penetration of cell layers by chemical compounds or other entities, and to assays for ascertaining the diffusion rate of members of a compound library through various confluent cell layers, e.g., endothelial or epithelial cell layers.
2. Background of the Invention
Pharmaceutical companies expend considerable resources on researching and developing drug therapies. The research and development process, from conception to eventual approval by the Food and Drug Administration (FDA), can last several years. Thus, in the initial stages, it is highly desirable to quickly rule out unusable chemical substances and focus efforts on effective substances.
In developing drug therapies, pharmaceutical companies typically start with a vast library of chemicals. From this library, a large number of the chemicals may have the potential to therapeutically act on the cells associated with the disease or ailment for which the drug is being developed. Determining which chemicals affect the cells is therefore an important step in drug development.
Chemotaxis is the directional movement (migration) of biological cells or organisms in response to concentration gradients of chemicals. Invasion is the movement (migration) of cells into or through a barrier. Tumor invasion is such action initiated by cancer cells into or through biological tissue in vivo, or, into or through extra cellular matrix proteins, e.g., collagen or matrigel, into or through barriers made of other substances, in vitro. Angiogenesis is the migration and formation of capillary blood vessels by endothelial cells. Growth is the increase in the size, form, or complexity of cells. Proliferation is growth of cells by cell division. Differentiation is the process by which cells change from a less specialized to a more specialized state usually associated with different functional roles and the expression of new and different traits. Interaction of cells is the alteration of cell behavior such as movement, invasion, angiogenesis, growth, proliferation, or differentiation in response to the presence and action of nearby cells of the same or different type.
The movement of compounds and structures within cells is another kind of cell activity that can be of interest in drug discovery. For example, powerful new optical detection systems can track the movement of florescent compounds (e.g., proteins) within the cell. Many different changes in internal cell activities in response to contact by compounds from a library with the cell's membrane and/or receptors can be observed with these new detection systems. These activities and similar activities are referred to herein collectively as “cell activity,” and the apparatus employed to perform the assays are referred to herein as “cell activity assay apparatus.”
One kind of single-site conventional cell activity assay apparatus referred to variously in the literature as “chemotaxis chambers,” “Boyden chambers,” “Boyden chemotaxis chambers,” “blind well chambers,” or “microchemotaxis chambers,” comprises two compartments separated by a membrane, with one or both of the compartments open to air. Multi-site apparatus are referred to as “multi-well chemotaxis chambers,” or “multi-well Boyden chambers,” and have the same basic site structure but have multiple sites. (See, e.g., U.S. Pat. Nos. 5,210,021 and 5,302,515.)
Assays employing this kind of apparatus pipette cells suspended in media into the upper compartments, and pipette chemotactic factors and controls into the bottom compartments. The chemotactic factors can be used in various dilutions to get a dose-response curve. The controls are generally of three kinds: (a) negative, when the same media that is used to suspend the cells is also used below the membrane, (b) chemokinetic, when a chemotactic factor is placed at the same concentration in the media with the cells and in the well on the opposite side of the membrane, and (c) positive, when a known chemoattractant is placed in the bottom wells. Chemokinetic controls allow the user to distinguish heightened random activity of the cells, due to contact with the chemotactic factor, from directional response in a concentration gradient of that chemotactic factor.
Cell activity assay apparatus can also be used to measure the response of cells of different origins—e.g., immune cells obtained from patients suffering from diseases—to a chemotactic factor of known chemotactic activity. In this case, the cells in question are interrogated by both a negative control and a known chemotactic factor to see if the differential response is depressed or normal.
Traditionally, chemotactic activity has been measured by establishing a stable concentration gradient in the cell activity assay apparatus; incubating it for a predetermined time; and then counting the cells that have migrated through the membrane (or into the membrane). A comparison is then made between the activity of the cells in a concentration gradient of the chemotactic factor being tested, and the activity of the cells in the absence of the concentration gradient.
In one type of conventional cell activity assay apparatus and method, the chemotactic response is measured by physically counting the number of cells on the membrane surface closest to the chamber containing the chemical agent. An example of this type of cell activity assay apparatus is described in U.S. Pat. No. 5,210,021 (Goodwin, Jr.), which is hereby incorporated by reference. One prior art method of obtaining quantitative data is to remove the membrane from the cell activity assay apparatus, remove the cells from the membrane surface closest to the chamber containing the original cell suspension, fix and stain the remaining cells, and then observe and count the stained cells under a microscope. Because of the time and expense associated with examining the entire membrane, only representative areas of the membrane are counted, rendering results less accurate than would otherwise be the case if the entire membrane were examined and counted.
Cell activity assays using a disposable ninety-six well microplate format, for example the ChemoTx™ System (available from Neuro Probe, Inc., Gaithersburg, Md.), are amenable to different methods of quantification of results. The manual staining and counting method described above can be used, but is not recommended due to the time involved. A preferred method is to centrifuge the microplate with the filter attached such that the cells that have migrated through the filter are deposited onto the bottom of the lower wells. The cells are then stained with MTT, MTS (available from Promega, Madison, Wis.), or a similar dye, and then read in a standard automated laboratory densitometric reader (sometimes referred to as an Elisa plate reader).
Another method of obtaining quantitative data with this apparatus is to dye the cells with a fluorescent material, e.g., Calcein AM (available from Molecular Probes, Eugene, Ore.); centrifuge the migrated cells into the microplate; and count cells with an automatic fluorescence plate reader (e.g., Cytofluor available from PE Biosystems, Foster City, Calif., Victor2 available from EG&G Wallac, Gaithersburg, Md., or fmax available from Molecular Devices, Sunnyvale, Calif.). The automatic plate reader excites the fluorescent dye in the migrated cells with one wavelength of light and reads the light emitted at a second wavelength. Alternatively, the cells that have not migrated are removed from the top of each site, and the plate with the framed membrane attached is read in the automatic fluorescent plate reader without spinning the cells into the plate, thereby counting the cells that have fallen off the filter into the lower well as well as those on the bottom of the membrane and in the pores of the membrane.
As described above, the past efforts at measuring chemotactic activity have focused on measuring or counting cells that have passed through a long, tortuous path, such as through a filter or thick membrane. Because of their dependence on cell migration, these techniques suffer from at least three significant drawbacks. First, the cells must migrate a considerable distance through the media to the chemotactic factor, which can add substantial time to the assay. Second, to obtain desirable (low) coefficients of variation, a relatively large number of cells is needed to calculate percentages of migration. Consequently, these assays demand large volumes of compound from a compound library, which are not always readily available. Third, in migration assays that count the number or percentage of cells that have passed through a filter, the results provide quantitative data, but not kinetic data. In addition, the results provide no information about the cells that have not passed through the filter.
Abbreviations & Definitions
1. “High throughput screening” is herein abbreviated to “HTS.”
2. “Cell-based high throughput screening” is herein abbreviated to “CBHTS.”
3. “High Content Screening” is herein abbreviated to “HCS.”
4. “Nanometer” is herein abbreviated to “nm.”
5. “Microliters” is herein abbreviated to “μl.”
6. “Micrograms” is herein abbreviated to “μg.”
7. “Cell activity assay apparatus” is herein abbreviated to “CAAA.”
8. “Test compound solution” is herein abbreviated to “TCS” and refers to a solution composed of a compound from a compound library dissolved in water, cell culture media, dimethyl sulfoxide (DMSO), other appropriate media, or a combination thereof.
9. “Cell suspension media” is herein abbreviated to “CSM” and refers to a media or fluid capable of suspending cells.
10. “Cell suspension” is herein abbreviated to “CS” and refers to a solution containing cells suspended in a cell suspension media.
11. “Point source” is herein abbreviated to “PS” and refers to a center area from which a chemical compound diffuses concentrically.